Manual WBC Count PDF

Manual WBC’s Count. A.Kewan 1  Objectives 1. Use the hematocytometer for WBC’s count. 2. Estimate the number of circulating WBC’s /mm³ of blood. 3. Understand the physiological and pathological conditions associated with the variation of WBC’s count. 4. List some fa...

Manual WBC’s Count. A.Kewan 1  Objectives 1. Use the hematocytometer for WBC’s count. 2. Estimate the number of circulating WBC’s /mm³ of blood. 3. Understand the physiological and pathological conditions associated with the variation of WBC’s count. 4. List some factors that cause source of error in WBC’s count. A.Kewan 2  Introduction Blood represent about 8% of total body weight. (5 – 6 liters). It consist of 1)) Cellular elements (45% of total blood volume) : Erythrocytes RBCs Leukocytes WBCs Platelets (thrombocytes) 2)) A complex liquid called plasma (55% of total blood volume). The cellular elements suspended in blood plasma A.Kewan 3 Blood composition A.Kewan 4  Introduction Blood performs many important functions within the body including: Supply of oxygen to tissues (bound to hemoglobin, which is carried in red cells). Supply of nutrients such as glucose, amino acid, and fatty acid (dissolved in the blood or bound to plasma proteins (e.g., blood lipids)). Removal of waste such as carbon dioxide, urea, and lactic acid. A.Kewan 5  Introduction Immunological functions, including circulation of white blood cells, and detection of foreign material by antibodies. Coagulation, which is one part of the body's self- repair mechanism (blood clotting after an open wound in order to stop bleeding). Messenger functions, including the transport of hormones and the signaling of tissue damage. Regulation of body pH. Regulation of core body temperature. A.Kewan 6  Introduction All various types of blood cells are produced in the bone marrow. A.Kewan 7  Manual WBC’s Count. WBC’s count : is the count of total # of leukocytes in a volume of blood. Expressed as WBC’s/1mm³ blood. WBC’s ,or leukocytes are almost colorless, nucleated cells. Leukocytes as well as erythrocytes are formed from stem cells in the bone marrow. They protect the body tissues by engulfing disease-bearing bacteria and foreign matter, by a process called phagocytosis. A.Kewan 8  Manual WBC’s Count. Normal range of WBC’s Newborn: 9000 – 30000 cells/mm³. Adult: 4000 – 11000 cells/mm³. - In this experiment you are going to determine the # of WBC’s in mm³ of blood. because this # is large, its practical to dilute a sample of blood with diluting solution (2% Glacial acetic acid with methylene blue) A.Kewan 9  Manual WBC’s Count. acetic acid will lyses all cytoplasmic membrane, therefore leaving the nuclei of WBC’s. methylene blue → coloring WBC’s nucleus. Count the # of WBC’s nucleus in a fraction of this diluted blood, and then multiplies by a correction factor. A.Kewan 10  Manual WBC’s Count. Materials 1. Blood sample. 2. WBC’s diluting pipette. 3. Diluting solution (2% Glacial acetic acid with methylene blue) 4. Hemocytometer. 5. Microscope. A.Kewan 11  Manual WBC’s Count. WBC’s pipette Consist of : - Stem. - Bulb(mixing chamber). It has 3 marks (0.5,1,and 11) and usually has white bead. A.Kewan 12 Manual WBC’s Count. A.Kewan 13 Manual WBC’s Count. A.Kewan 14 Manual WBC’s Count. A.Kewan 15  Manual WBC’s Count. The Hemocytometer : - Contains two square ruled areas separated by an h-shaped moat. - Depth = 0.1mm. - Each square has total area of 9 mm². - These squares are divided into 9 primary squares with an area of 1mm² for each one of them. - The four corner primary squares are used when counting WBC’s. A.Kewan 16  Manual WBC’s Count. - Each one of these 4 large corner squares, contain 16 smaller secondary (medium) squares. - The central primary square is used for RBC’s count. its consist of 25 secondary (medium) squares. - Each of these 25 square is further divided into 16 smaller tertiary ( smallest ) squares. A.Kewan 17  Manual WBC’s Count. Procedure: With a safety bulb draw blood up to 0.5 marks on WBC’s pipette and complete to 11 with WBC’s diluting solution (2% Glacial acetic acid with methylene blue). 2. Mix for 2-3 minute. A.Kewan 18  Manual WBC’s Count. 3. Charge hemocytometer : load the counting chamber with diluted blood as follows: Discard the first 4-5 drops. Place tip of the pipette at edge of the central platform of hemocytometer slide and let a drop of diluted blood run between the hemocyto- meter slide and cover slip by capillary. A.Kewan 19  Manual WBC’s Count. 4. Let the hemocytometer to stand on the bench for 3-5 minutes, so the cells are settled down. A.Kewan 20  Manual WBC’s Count. 5. Count and calculate: 5.1 WBC’s count is performed with neubauer hemocytometer as follow: a- Place the hemocytometer on the microscope stage and focus with 4X objective, on the ruled area move to the 10X and focus on one of the large corner square which is divided into 16 medium squares. A.Kewan 21 A.Kewan 22 A.Kewan 23  Manual WBC’s Count. B- count the cells in each large squares starting from the upper left medium square and follows the pattern indicated by the arrow in the next figure. C- count all cells that touch any of the upper and left lines, do not count any cells that touches a lower or right line. A.Kewan 24 Manual WBC’s Count. A.Kewan 25 A.Kewan 26 A.Kewan 27  Manual WBC’s Count. D- get the total # of cells counted in all 4 large squares. Variation between two W squares shouldn’t be more than 10 cells. A.Kewan 28  calculation WBC’s count : is the count of total # of leukocytes in 1mm³ of undiluted blood. But we counted WBC’s in a volume less than 1mm³ of diluted blood. So, we need two correction factor. A.Kewan 29  Manual WBC’s Count. B- Calculation: Number of WBCs/mm³ of undiluted blood = counted cells in 4 large squares X diluting correction factor X volume correction factor. The dilution factor = total volume / sample volume. DF = (11 – 1) / 0.5 = 20 Volume correction factor = Desired volume / counted volume in 4 squares. VCF = 1 mm³ / counted volume A.Kewan 30  Manual WBC’s Count. counted volume = The total volume of the 4 large squares = Volume x # of large squares = (width x length x depth )x 4 = (1mm x 1mm x 1/10 mm) x 4 = 0.4 VCF = 1 mm³ / counted volume = 1 / 0.4 = 2.5 A.Kewan 31  Manual WBC’s Count. Example: If total # of WBC’s in 4 large squares is 120 Then the # of WBC’s in1mm³= 120 x 20 x 2.5 = 120 x 50 = 6000 cells So, number of cells/mm³ = n x 20 x 2.5 = n x 50 A.Kewan 32  Manual WBC’s Count. Significance of the test : Leukocytosis: a condition when the total # of WBC’s exceeds normal range (11000 cells/mm³) Occur as indicator of: - body defense against foreign materials (bacteria, parasites, toxins). - Metabolic disorders. - Chemical and drug poisoning. - Acute hemorrhage. A.Kewan 33  Manual WBC’s Count. Leukopenia: a condition when the total # of WBC’s below normal range (4000 cells/mm³). Result from: ‫ ــ‬X-ray therapy. – Alcoholism. – Antibiotic therapy. – Typhoid infection. – Measles. – Infectious hepatitis. – Tuberculosis (TB). – Cirrhosis of the liver. A.Kewan 34  Manual WBC’s Count. Sources of error 1. Flooding of chamber with excess sample. 2. Failing to count all the cells in the squares or conversely including artifacts in the count. 3. when the blood diluted too much, or concentrated too much. A.Kewan 35  https://www.youtube.com/watch?v=xyhbIP SLBsA https://www.youtube.com/watch?v=s- Zt9jCZyDQ https://www.youtube.com/watch?v=sCgMl 9eHZxU A.Kewan 36